The long term objective of this research is a detailed understanding on the DNA binding of model ligands at the molecular level, through which rational designs of effective anticancer and antiviral drugs can be based. This proposal focuses on a detailed sequence-specific and structural elucidation of actinomycin D (ACTD) binding to single- and double-stranded DNA oligonucleotides containing no canonical GpC sites, utilizing a novel binding mode involving possibly base displacements. Detailed understanding on the workings of this new binding mode will greatly expand the repertoire of this supposedly well understood drug and may have important implication on its role as a transcription inhibitor and for the design of other sequencespecific drugs. Our immediate goals for the next four years will be: (1) To demonstrate that ACTD binds strongly to a general sequence motif of d(vGwCxyzG) and to provide rationales for such a binding, likely a combination of monomeric hairpin and slipped dimeric duplex, with the 3'-sides of the two G bases stacking on the opposite faces of the drug chromophore with likely base displacements. (2) To further delineate these binding modes by carrying out systematic studies with DNA oligomers of apparent single-stranded or duplex-prone sequence motifs. Furthermore, to re-investigate the previously observed unusually slow association kinetics of ACTD binding to d(CATGGCCATG)2 and to rationalize the results in terms of drug-induced duplex denaturation and subsequent binding via the speculated mode. (3) To carry out ACTD binding studies with the corresponding GC-containing oligomers to see how their binding and spectral characteristics differ from those of the GTC- or GAC-containing counterparts, and to demonstrate that the new binding mode may also be operative in these GC-containing oligomers and may, in fact, be more important than the classic binding mode in some sequence contexts. (4) To carry out NMR studies through collaborations with other laboratories to obtain detailed structural information on drug-DNA complexes so as to test the validity of our proposed dimeric duplex and monomeric hairpin binding models. The knowledge gained on the structural basis of these binding modes of ACTD will assist in the design of new classes of sequence specific antitumor agents. These goals are to be achieved via systematic spectral titrations (absorbance, circular dichroic, fluorescence, and NMR), association and dissociation kinetic measurements, optical and calorimetric melting experiments, and capillary electrophoretic migration analyses. DNA oligonucleotides of appropriate lengths and sequences will be used in these studies.